Report for 9/8/2016 through 8/17/2016: Over 2016-17 Feldman engaged in research projects with six labs: three from NICHD, one from NIEHS, one from NINR and one from the Childrens National Medical Center. Porter Lab (NICHD): Genetic Dissection and Creation of Human Disease Models of Sterol Metabolism. Smith-Lemli-Opitz Syndrome (SLOS) is an autosomal recessive, multiple malformation syndrome with pediatric onset characterized by intellectual disability and aberrant behavior. Phenotypic characterization is ongoing of zebrafish carrying mutant alleles of dhcr7, the zebrafish ortholog to the human SLOS gene DHCR7, which were generated with support from the Core in previous years. This project will continue in 2017-18. The Core also used Crispr-Cas9 technology to create additional genetic mutant lines for the Porter labs in genes with roles in other steps in cholesterol metabolism. Phenotypic characterization by the Porter is ongoing and will continue into 2017-18. Stratakis Lab (NICHD): Function of Zebrafish Orthologs to Human Genes Implicated in Disorders of the Pituitary-Adrenal Axis. (1) Gigantism arises due to excess growth hormone (GH) secretion during childhood, before the growth plates close. Since 2012, the Core has supported this labs investigation of the zebrafish ortholog to a human gene implicated as a driver of gigantism. The lab published a paper in June 2016, with Feldman as co-author, which included a description of this genes developmental expression in zebrafish. This year, the Stratakis lab also began to test the effect on growth and development of zebrafish in which this gene is chronically overexpressed in tissue-specific or ubiquitous locations, using the Gal4/UAS transgene system. These studies will continue in 2017-18. Last year the Core used Crispr-Cas9 methods to generate for the Stratakis lab zebrafish carrying loss-of-function mutations in the above gene and three other zebrafish orthologs to genes implicated human growth anomalies. Characterization of the resulting phenotypes is ongoing and will continue intop 2017-18. (2) Since 2012, the Core has also supported this labs investigation of the function of two zebrafish orthologs to human adrenal hyperplasia and Cushing disease-associated genes. Over previous years, the Core helped this lab generate and acquire, respectively, zebrafish carrying loss-of-function mutation for each of these orthologs. Phenotypic characterization has found notable effects on juvenile growth in the case of one gene and on early embryogenesis in the case of the other. Phenotypic characterization of these lines will continue into 2017-18. This year we generated mutants for six new genes whose human orthologs are implicated in adrenal hyperplasia and/or Cushing disease and phenotypic characterization of these mutations will commence in 2017-18. Kaler Lab (NICHD): Modeling Copper Deficiency-Associated Distal Motoneuropathy The Menkes gene encodes ATP7A, a copper-binding ATPase localized to the plasma membrane and the trans-Golgi network (TGN), which is critical for proper intracellular copper distribution. Complete loss of ATP7A causes Menkes disease causes a severe human disease leading to childhood death unless early intervention with copper therapy is performed. Two ATP7A missense mutations cause a milder syndrome than Menkes, a distal motoneuropathy that is nevertheless debilitating to children and young adults. Since 2013, the Core has supported a project to clarify the structure-function relationship of ATP7A and motor neuron defects from the perspective of these missense mutations. Over previous years the Core supported the Kaler labs work to visualize and compare motor neuron growth during embryogenesis of WT zebrafish embryos and embryos homozygous for null mutations in their ATP7A ortholog: atp7a. This comparison will continue into 2017-18. In parallel to these studies, Feldman and Tsai-Morris have made a concerted effort to establish genome-editing technology in the Core, using CRISPR-Cas9 technology in combination with donor DNA and comparing two general approaches; double-stranded donor DNA for homologous-recombination-based genome editing and single-stranded DNA for homology-directed repair-based genome editing. The initial goal is to induce formation of zebrafish atp7a point mutations cognate to one of the human ATP7a motoneuropathy alleles. This work will continue into 2017-18. Blackshear lab (NIEHS): Assessing Functions of a Zinc-Finger Protein Gene Family in Zebrafish Hematopoesis.The Blackshear lab is interested in dissecting the connections of a family of zinc-finger proteins and blood development. They contracted the NICHD Zebrafish Core to create null mutations in each of the seven zebrafish orthologues to this family, determine the viability or fertility of such mutants and if viable, to provide blood samples from mutants for the Blackshear lab to analyze. This year we created null alleles for six out of the seven genes. Phenotypic analysis has commenced, with no aberrations in homozygous null fish yet identified for three of the seven genes that have been tested. Phenotypic analysis will continue into 2017-18. Meilleur lab (NINR): Testing the Ability of Small Molecules to Mitigate Myopathy in Zebrafish ryr1b Mutants. This new project will begin in earnest in 2017-18, but we have already helped formulate a plan with Dr. Meilleur and colleagues to test candidate drugs they are currently identifying for their ability to potentially ameliorate muscle defects seen in zebrafish mutants that carry mutations in a gene, ryr1b, for which mutations in its human counterpart are implicated in various myopathies. We have acquired larvae carrying the ryr1b mutation from an outside source and we are currently raising them to adulthood. Tuchman Lab (Childrens National Medical Center): Finding Neuroprotective Drugs to Mitigate Hyperammonemia, a Consequence of Urea Cycle Defects & Liver Failure. Exposure of the brain to high ammonia causes neurcognitive deficits, intellectual disabilities, coma and death. Since 2012, the Core has helped this lab to use zebrafish embryos to identify small molecules able to diminish the effects of hyperammonemia. Over previous years, a library of hundreds of small molecules with known safety profiles for humans was screened and several promising candidates were identified for follow-up validation studies in zebrafish and other animal models. Last year, the Core supported the Tuchman lab increase throughput of this screen, bolstered by additional personnel from the Tuchman lab, and the Cores implementation of NICHDs massive embryo production systems (MEPS; see above) as a source for embryos, enabling them to complete a screen of an additional 10,000 compounds in 2016-17. Additional candidate compounds were thus identified and secondary screens and dose-response studies on lead compounds will continue in 2017-18.